Abstract A39: Epigenetic upregulation of lncRNA HOTAIR expression in three-dimensional culture of triple-negative breast cancer cells

Abstract

Abstract lncRNA HOX transcript antisense RNA (HOTAIR) represses gene expression via recruitment of the chromatin modifying complex polycomb repressive complex 2 (PRC2) and its overexpression mediates invasion and metastasis in breast cancer. In an apparent paradox we observed silencing of HOTAIR expression in MDA-MB231 cells, an invasive and metastatic triple-negative breast cancer cell line in two dimensional monolayer culture (2-D). This observation prompted us to question limitations of 2-D culture as a model system to investigate the role of HOTAIR in breast cancer. Thus we examined HOTAIR expression in reconstituted basement membrane matrix Matrigel-based three dimensional organotypic culture (rBM 3-D) because MDA-MB231 cells undergo stellate morphogenesis, a phenomenon that is characteristic of invasive cancer cells. In MDA-MB231 cells HOTAIR expression in rBM 3-D culture and the grafted tumors exhibited ~130-fold and ~60-fold over than that in 2-D culture, respectively. Induction of HOTAIR in rBM 3-D culture involved transcription from an uncharacterized promoter located in the 1st intron of the HOXC11 gene. However HOXC11 did not exhibit difference in its expression between 2-D and rBM 3-D cultures. Induction of HOTAIR in rBM 3-D culture was congruent to a wide spread increase in expression of the protein coding HOX genes. Moreover stellate morphogenesis was abrogated by the HOTAIR-specific siRNA and replaced by mass morphogenesis that is characteristic of non-invasive cells. To validate induction of HOTAIR as a response to extracellular matrix (ECM), we knocked down a major ECM receptor integrin α2 using lentiviral vectors expressing integrin α2-specific shRNA. As expected knockdown of integrin α2 resulted in a substantial decrease of HOTAIR expression. Because the HOX genes are tightly regulated by CpG methylation, we compared CpG methylation of the promoters of HOTAIR and HOXA9 in 2-D and rBM 3-D cultures. Indeed the promoters of HOTAIR and HOXA9 exhibited lower CpG methylation in rBM 3-D culture than that in 2-D culture. Moreover 5-Azacytidinewe up-regulated the expression of HOTAIR and HOXA9 in 2-D culture of MDA-mb231 cells. Moreover induction of HOTAIR was associated with increased binding of BRD4 to the HOTAIR promoter region located in the HOXC11 1st intron. The BRD4-specific siRNA substantially reduced the expression of HOTAIR in rBM 3-D culture. Taken together our findings suggest that ECM up-regulate the expression of HOTAIR via reduced CpG methylation and increased BRD4 binding in a human triple-negative breast cancer cell line. Note: This abstract was not presented at the conference. Citation Format: Miao Li, Xi Li, Bin Shan. Epigenetic upregulation of lncRNA HOTAIR expression in three-dimensional culture of triple-negative breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A39.